Mechanisms of Islet Beta Cell Dysfunction in Diabetes

项目摘要 鞘脂神经酰胺[CER]已被证明是信号转导的重要介质 过程导致多种细胞反应，包括凋亡。尽管令人信服的实验 有证据表明，CER依赖的信号传导机制可能是体外细胞功能障碍的基础， 以及胰岛素分泌受损的体内模型，关于确切的作用模式知之甚少 CER在导致胰岛细胞代谢失调的信号事件中的作用。我们的初步研究结果 表明INS 832/13细胞和原代大鼠胰岛长期暴露于升高葡萄糖和脂质 促进冈田酸敏感性蛋白磷酸酶[CAPP]的CER依赖性活化， 吞噬NADPH氧化酶[NOX]导致线粒体失调。我们还提出了初步的 有证据表明这两种途径在来自Zucker糖尿病脂肪[ZDF]大鼠的胰岛中被加速， 一种被广泛接受的2型糖尿病模型。 基于这些数据，我们假设，细胞内CER的积累，诱导以下慢性 将分离的细胞暴露于葡萄糖和脂质，引起线粒体功能障碍，导致细胞死亡。的 拟议研究的三个具体目的是：[I]证明糖脂毒性条件促进 CER介导的CAPP线粒体亚型的活化导致去磷酸化和失活 Bcl-2最终导致胰岛细胞的线粒体功能障碍;[II]以证明糖脂毒性 条件促进CER介导的全酶组装和NOX的功能活化，从而导致 ROS的产生和胰岛细胞的线粒体功能障碍的相关发作;和[III]至 精确定义线粒体缺陷和代谢功能障碍的进展和预防[确定] 根据目的I和II]通过CER合成抑制剂在ZDF大鼠胰岛中的作用。我们将使用一些生化的， 分子生物学、细胞生物学和免疫学方法来验证我们的假设， 我们在INS 832/13细胞、原代大鼠胰岛和整个动物中的目标。希望来自于 拟议的研究将提供新的见解，具体的CER敏感信号步骤的调节作用， 线粒体功能障碍的发生导致胰岛细胞在压力下死亡， 糖脂毒性疾病。我们的长期目标是开发特定的治疗方式，以防止 这些细胞缺陷的建立和糖尿病的发病。 我们提出的研究与VA研究和患者护理任务直接相关。可用数据 这清楚地表明，退伍军人比普通人群更容易患糖尿病，这是主要的糖尿病之一。 与肥胖相关的并发症。根据美国糖尿病协会，超过7%的 美国人患有糖尿病，而且发病率随着年龄的增长而增加。在接受退伍军人事务部医疗保健的退伍军人中， 平均年龄比一般人口大的人，这一比率超过20%。根据VA，70% 在通过该部门获得健康福利的750万退伍军人中，有五分之一的人肥胖， 糖尿病会导致心脏病、高血压和截肢。我们设想， 将为特定CER敏感信号的调节作用提供新的见解 线粒体功能障碍的发生步骤导致细胞在胁迫下死亡， 糖脂毒性状况。从我们的研究中积累的数据可能成为发展的基础。 具体的治疗方式，以防止建立这些细胞缺陷和糖尿病的发病。

Project Summary The sphingolipid ceramide [CER] has been shown to be an important mediator of signal transduction processes leading to a variety of cellular responses, including apoptosis. Despite the compelling experimental evidence to suggest that CER-dependent signaling mechanisms might underlie -cell dysfunction in in vitro and in vivo models of impaired insulin secretion, very little is known with regard to the precise modes of action of CER in the signaling events leading to metabolic dysregulation of the islet -cell. Our preliminary findings suggest that long-term exposure of INS 832/13 cells and primary rat islets to elevated glucose and lipids promote CER-dependent activation of an okadaic acid-sensitive protein phosphatase [CAPP] and the phagocytic NADPH-oxidase [NOX] leading to mitochondrial dysregulation. We also present preliminary evidence to indicate that these two pathways are accelerated in islets from the Zucker Diabetic Fatty [ZDF] rat, a widely accepted model for type 2 diabetes. Based on these data we hypothesize that an accumulation of intracellular CER, induced following chronic exposure of isolated -cells to glucose and lipids, causes mitochondrial dysfunction leading to cell demise. The three Specific Aims of the proposed studies are: [I] to demonstrate that glucolipotoxic conditions promote CER-mediated activation of the mitochondrial isoform of CAPP leading to dephosphorylation and inactivation of Bcl-2 culminating in the mitochondrial dysfunction of the islet -cell; [II] to demonstrate that glucolipotoxic conditions promote CER-mediated holoenzyme assembly and functional activation of NOX to result in the generation of ROS and the associated onset of mitochondrial dysfunction of the islet -cell; and [III] to precisely define the progression, and prevention of mitochondrial defects and metabolic dysfunction [identified under Aims I and II] by CER synthesis inhibitors in the ZDF rat islet. We will employ a number of biochemical, molecular biological, cell biological and immunological approaches to validate our hypothesis and accomplish our goals in INS 832/13 cells, primary rat islets and whole animals. It is hoped that data derived from the proposed studies will provide fresh insights into the regulatory roles of specific CER-sensitive signaling steps in the onset of mitochondrial dysfunction leading to the demise of the islet -cell under the duress of glucolipotoxic conditions. Our long-term goal is to develop specific therapeutic modalities to prevent the establishment of these cell defects and the onset of diabetes. Our proposed studies have direct relevance to the VA research and patient care missions. Available data clearly suggest that veterans are more likely than the general population to have diabetes, one of the major complications associated with obesity. According to the American Diabetes Association, greater than 7% of the U.S. population has diabetes, and the rate increases with age. Among veterans receiving VA health care, who are on average older than the general population, the rate is greater than 20%. According to the VA, 70% of the 7.5 million veterans who receive health benefits through the department are obese, and one in five has diabetes, which can lead to heart disease, high blood pressure and amputations. We envision that data derived from the proposed studies will provide fresh insights into regulatory roles of specific CER-sensitive signaling steps in the onset of mitochondrial dysfunction leading to the demise of the -cell under the duress of glucolipotoxic conditions. The data accrued from our studies might form the basis for the development of specific therapeutic modalities to prevent the establishment of these -cell defects and the onset of diabetes.